The present invention relates in general to karyotyping via fluorescent in situ hybridization. More particularly, the present invention relates to a method of and a composite for fluorescent in situ hybridization using a hybridization composite of chromosomal paints which is free of added unlabeled highly repetitive DNA, such as Cot-I fraction, and which is optionally designed to maximize denaturation and hybridization between DNA probes and target DNAs in cellular samples, thereby enabling reduction of about at least 10-200 fold in concentration of probe employed.
The genome of higher organisms, such as human beings, include a large fraction of repetitive sequences which are spread among all chromosomes. As learned from Cot curves, the human genome may roughly be categorized into (i) highly repetitive sequences, also known as the fast component or the first fraction to reassociate, or Cot-I fraction (Cot=ca. 10.sup.-4 M.multidot.sec-ca. 2.multidot.10.sup.-2 M.multidot.sec, Cot.sub.1/2 =ca. 1.3.multidot.10.sup.-3 M.multidot.sec), such as, but not limited to, Alu and Alu-like sequences, LINE 1 and 2, sequences, .alpha. satellite sequences, .beta. satellite sequences, classical satellite sequences, etc.; (ii) intermediate repetitive sequences, also known as the intermediate component (Cot=ca. 2.multidot.10.sup.-1 M.multidot.sec-ca. 10.sup.2 M.multidot.sec, Cot.sub.1/2 =ca. 1.9 M.multidot.sec), such as, but not limited to, ribosomal DNA; and (iii) low repetitive and unique sequences, also known as the slow component, or the last fraction to renature (Cot=ca. 10.sup.2 M.multidot.sec-ca. 10.sup.4 M.multidot.sec, Cot.sub.1/2 =ca. 6.3.multidot.10.sup.2 M.multidot.sec), such as, but not limited to, gene encoding sequences. Additional details concerning these fractions of the human genome are described in, for example, Genes V, Benjamin Lewin, Oxford University Press. (1994) pp. 660-675, which is incorporated by reference as if fully set forth herein.
Therefore, when a probe including repetitive sequences, such as a chromosomal paint, is hybridized with target DNA also including repetitive sequences, such as a chromosome spread, measures must be taken to limit or reduce hybridization of the labeled repetitive sequences from the probe with the repetitive sequences in the target DNA.
These measures may include one or more of the following: (i) removal of the repetitive sequences from the probe; (ii) blocking the repetitive sequences of the probe by addition of unlabeled repetitive sequences in excess to the probe composite and providing annealing conditions underwhich substantially only repetitive sequences hybridize, typically by limiting hybridization time; and/or (iii) blocking the repetitive sequences of the target DNA by addition of unlabeled repetitive sequences in excess to the target DNA during the prehybridization procedure. When fluorescent in situ hybridization is envisaged, blocking the repetitive sequences of the probe is currently accepted as the method of choice, yielding best results.
However, all of these methods call for additional time consuming steps and must be executed very precisely for good results.
The use of fluorescent dyes (i.e., fluorescent probes, fluorophores, fluorochromes), is one of the most powerful and common tools for analyzing tissues and cells. Fluorescence microscopy is therefore one of the most important experimental methods used in light microscopy [Lakowicz (1983) Principles of fluorescence spectroscopy, Plenum Press, New York, London].
The power of fluorescent probes, is mainly due to the great variety of biological structures to which specific dyes can be bound [Waggoner (1986) Applications of fluorescence in the biomedical sciences, Eds. Taylor et al., New York: Alan R. Liss, Inc. pp. 3-28]. For a detailed review of fluorescent probes see, Mason, editor (1993) Fluorescent and Luminescent Probes for Biological Activity, Biological Techniques Series, edited by Sattelle, Academic Press Limited, London; and, Ploem and Tanke (1987) Introduction to Fluorescence Microscopy, Oxford University Press, Royal Microscopical Society.
The rapid development of new and more sophisticated fluorescent dye molecules continues to create a need for more advanced fluorescence imaging techniques that can utilize the full potential of these dyes. For a discussion of the revolutionary impact fluorescent dyes have had, and will continue to have, on the way research is conducted today, refer to Taylor et al. (1992) The New Vision of Light Microscopy, American Scientist, Vol. 80, pp. 322-335.
An important example where the detection of multiple fluorescent probes can be of significant advantage is FISH (fluorescent in situ hybridization) [Emanuel (1993) Growth Genetics and Hormones 9, pp. 6-12], which is used to analyze genes at the chromosome level, and find possible genetic defects such as gene/chromosome amplification, deletion, translocation, rearrangement and other abnormalities associated with genes and/or chromosomes.
Certain diseases and disorders, including many cancers and birth defects, are genetic disorders caused by defects (i.e., mutations) in one or more genes. Many other diseases arc known or believed to have a genetic component(s), that is, there exists a genetic defect(s) that does not alone cause the disease but contributes to it, or increases the probability of developing the disease later in life, phenomena known in the art as multifactorial diseases and genetic predispositions.
Correlation of visible genetic defects with known diseases would allow physicians to make definitive diagnoses, and permit early detection and treatment of many diseases. Genetic counseling could alert prospective parents and at-risk individuals to the possibility of potentially serious medical problems in the future, permitting appropriate intervention.
More than 8,000 genetic disorders have now been identified, many of which are associated with multiple genetic defects. Following the discovery that chromosomes are the carriers of hereditary information, scientists reasoned that it should be possible to document visible defects in chromosomes that were responsible for specific disorders.
In the 1960's, staining techniques were developed for microscopy-based classification of metaphase chromosomes spreads. For several decades, visual analysis of chromosomes banding patterns has been used to correlate human genetic disorders with observed structural abnormalities in metaphase chromosomes. Chromosomes are typically examined by brightfield microscopy after Giemsa staining (G-banding), or by fluorescence microscopy after fluorescence staining (R-banding), to reveal characteristic light and dark bands along their length. Careful comparison of a patient's banding pattern with those of normal chromosomes can reveal abnormalities such as translocations (exchange of genetic material between or within chromosomes), deletions (missing chromosome(s) or fragment(s) thereof), additions, inversions and other defects that cause deformities and genetic diseases. Yet conventional chromosome banding techniques are limited in resolution.
Fluorescent in situ hybridization (FISH) has evolved over the past 25 years through the improvement of a number of complementary techniques. Its emergence has been driven by the desire of cytogeneticists to develop better tools for mapping the precise location of genes on chromosomes, and to detect very small genetic defects not visible by gross staining of chromosomes.
The human genome project (HGP), a bold initiative to identify and map all human genes, has identified interest in FISH and has hastened the development of much-needed DNA probes. Current FISH techniques have also been made possible by the concurrent development of powerful immunological probes, a growing variety of excellent fluorescent dyes for microscopy and spectroscopy, and dramatic improvements in the objectives, illuminators and filters used for fluorescence microscopy.
The power and utility of FISH is due to many factors: (i) FISH can be used not only on isolated chromosomes and nucleus, but also whole cells within fixed, paraffin-embedded tissue sections; (ii) it can detect relatively small defects (ability of detecting smaller defects constantly increases); (iii) it can provide results relatively fast; (iv) its moderate cost allows it to be used in most diagnostic and research laboratories; (v) adaptation can be developed for various probes and specimen types; and, (vi) high specificity and sensitivity can be achieved; (vii) within a short time, in some cases in the range of two hours.
Many FISH applications merely require from the cytogeneticist to look through the eyepieces of a microscope, or at the image on the monitor, and to determine whether a fluorescent label is present or absent. With somewhat more complex specimens, a simple count of one or two colored labels may be done. However, the ability to process digital images and extract numerical data from them adds a vast new set of capabilities to FISH techniques.
An appropriate imaging method, can enhance very faint FISH images so that labeled chromosomes and loci are clearly identifiable. Under readily achieved experimental conditions, the number of labeled sites can be automatically counted. In addition, the intensity at each labeled site can be measured and the amount of DNA calculated to reveal, for example, the number of copies present of a particular gene.
As discussed above, FISH can provide information on the location of the labeled probe, the number of labeled sites on each chromosome, and the intensity of labeling (the amount of genetic material) at each site. Centromeric (repetitive DNA) probes and chromosome paints are used to tag, count the number of copies present of each targeted chromosome and detect structural changes. Locus-specific probes are used to map the location of small regions of genetic material. These types of probes can be used on intact interphase nucleus as well as metaphase chromosome spreads, and can be counted visually or automatically by a suitable algorithm. They are routinely used to identify genetic diseases characterized by numerical and/or structural changes in chromosomes and/or gene.
Using FISH it is possible to uniformly label the entire surface of one specific chromosome by isolating the chromosome (using flow cytometry, for example), physically (e.g., by sonication) or enzymatically (e.g., by endonucleases) chopping it up, and generating a set of probes against all of the fragments. Whole chromosome probes, also known as chromosome paints, fluorescently label all copies of their target chromosome. One important application of chromosome painting is the detection of translocation of genetic material between two chromosomes, as characteristically occurs in early stages of certain cancers, yet other chromosome aberrations are also detectable.
For example, if chromosome A is specifically labeled with a green paint and chromosome B is labeled with a red paint, translocation of genetic material from A to B will appear as a green area juxtaposed to a red area (and vice versa).
Typically, chromosome paints generated from normal chromosomes are used to detect structural aberrations associated with abnormal (patient) chromosomes. Reverse chromosome painting uses probes generated from an abnormal chromosome to identify DNA from various normal chromosomes which contributed material to the abnormal chromosome.
The method of the present invention, as exemplified hereinbelow in the Examples section, enables to paint the 24 different chromosomes comprising the human karyotype (i.e., genome), each in a different color, and simultaneously detect, identify and meaningfully display a color human karyotype, using a single hybridization procedure followed by a single short measurement.
A remarked improvement in multicolor fluorescent dyes used for labeling chromosome paints is the introduction of combinatorial fluorescent strategies (e.g., combinatorial labeling and combinatorial hybridization) which employ various combinations of few basic fluorescent dyes. For further detail concerning combinatorial hybridization see, Ried et al., (1992) Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. USA 89, 1388-1392; and, Ried (Jan. 1994) Fluoreszenz in situ Hybridizierung in der genetischen Diagnostik, Faculty of theoretical medicine, Ruprecht-Karls University Heidelberg, both are incorporated by reference as if fully set forth herein. For further detail concerning combinatorial labeling see du-Manoir et al. (1993) Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum. Genet. 90, 590-610, and U.S. Pat. No. 5,759,781 to Ward et al., both are incorporated by reference as if fully set forth herein.
Numerous methods are available to label DNA probes for use in FISH (including chromosome painting) assays, including indirect methods whereby a hapten such as biotin or digoxigenin is incorporated into DNA using enzymatic reactions. Following hybridization to a metaphase chromosome spread or interphase nucleus, a fluorescent label is attached to the hybrid through the use of immunological methods. More recently, fluorescent dyes have been directly incorporated into probes and detected without the use of an intermediate step. Standard FISH dyes include fluorescein, rhodamine, Texas-Red and cascade blue, and multiprobe FISH analysis can be accomplished by labeling different probes with different haptens or fluorescent dyes and combinations thereof, known in the art as combinatorial labeling. Alternatively, a pool of a given probe may be divided into sub-pools, each labeled with a different fluorophore, after which the sub-pools are regrouped to yield otherwise similar hybridization results, a method known in the art as combinatorial hybridization. According to both labeling strategies obtained are combinatorial probes. Thus, when any of the terms "combination of fluorophores" is used herein in this document and especially in the claims below, it refers both to combinatorial labeling and to combinatorial hybridization, as described above.
The use of combinatorial fluorophores for chromosome painting and karyotyping, multicolor chromosome banding and comparative genome hybridization is described in details in U.S. Pat. No. 5,817,462 in U.S. Pat. No. 5,759,781 to Ward et al. and in Science magazine [E. Schroeck et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science, 273, 494-497], all are incorporated by reference as if fully set forth herein.
The main progress described in Science is that whole genome scanning by spectral imaging allows instantaneous visualization of defined emission spectra for each human chromosome after fluorescence in situ hybridization (FISH). By means of computer separation (classification) of spectra, spectrally-overlapping chromosome-specific DNA probes are resolved and all human chromosomes are simultaneously identified.
This spectral imaging approach therein described combines Fourier spectroscopy, charge coupled device (CCD)-imaging, and optical microscopy to measure simultaneously at all points in the sample emission spectra in the visible and near-infrared spectral range. This allows the use of multiple spectrally overlapping probes. The approach is based on the measurement of a discrete spectrum (identified from a sequence of intensities at every pixel measured at many different wavelengths), which facilitates the discrimination of multiple fluorophores. In dramatic contrast to conventional epifluorescence microscopy in which fluorochrome discrimination is based on the measurement of the fluorescence intensity through a small number of narrow band fluorochrome specific optical filters [see, Speicher et al. (1996) Nature Genetics. 12:368-375], the use of spectral karyotyping, as therein described, allows all the available information present in the emitted photons within the spectrum of emitted light, to be used for analysis.
The spectral-based method for discriminating spectrally overlapping fluorophores (classification) is readily extended to a large number of fluorochromes, provided there are measurable and consistent differences in the emission spectrum of each fluorochrome.
Simultaneous identification of each human chromosome in metaphase preparations, an approach referred to as spectral karyotyping, is also reported. To this end, chromosome-specific composite libraries generated by polymerase chain reaction (PCR) from flow-sorted human chromosomes are directly labeled with nucleotides conjugated to five different fluorophores or combinations thereof A composite probe set containing all 24 chromosomes is then hybridized to metaphase chromosomes. Chromosome-specific labeling is achieved by suppression hybridization. Specifically, repetitive sequences in the hybridization composite are blocked by the addition of an excess of unlabeled human Cot-I DNA.
The hybridization is presented in both RGB display and classification colors. Display colors allow all human chromosomes to be readily visualized after spectral imaging, and based on spectral measurements at each pixel, a chromosome classification algorithm is applied to spectrally karyotype all human chromosomes. One of the most important analysis algorithms is the spectral-based classification algorithm that enables multiple different spectra in the image to be identified and highlighted in classification-colors. This allows assignment of a specific classification-color to all human chromosomes based on their spectra. This algorithm assumes that the (reference) spectrum of each chromosome has been measured and stored in a reference library in the computer. A distinguishing classification-color is assigned to each pixel in the image according to the classification-color assigned to the reference spectrum that is most similar to the spectrum at that given pixel. A minimal square error algorithm as shown in Equation 1: ##EQU1## is computed for every pixel, in which I.sub.x.v (.lambda.) is the normalized spectrum at pixel coordinates x,y and I.sub.n (.lambda.) represents the normalized reference spectrum for each of the chromosome n=1, 2, . . . , 23, 24. After calculating the value of S.sub.x.v.n for all reference spectra, the smallest value is chosen and an artificial classification-color is assigned to that pixel in accordance with the classification-color assigned to the most similar reference spectrum.
The potential of spectral karyotyping as a screening method for chromosomal aberrations was further explored by analyzing clinical samples from multiple laboratories where conventional banding methods or FISH with chromosome painting probes had been previously performed. In all cases, G-banding and spectral karyotyping revealed consistent results. In some cases, Giemsa-banding was not sufficient to entirely interpret the chromosomal aberrations. In these cases, the diagnosis of chromosomal aberrations by spectral karyotyping was confirmed with conventional dual-color FISH analysis. The smallest discernible aberration analyzed for this report was a translocation t(1;11)(q44;p15.3) in which the reciprocal translocation was unrecognizable by conventional banding analysis. The origin of the chromosomal material that contributed to the reciprocal translocation was correctly classified. The translocated segments on chromosomes 1 and 11 had been confirmed by subtelomere specific cosmid probes for chromosomes 1q and 11p. On the basis of the location of the probes utilized, the size of the alteration was estimated to be &gt;1,500 kbp. In a second case, banding analysis suggested a translocation of a segment of chromosome 4 to chromosome 12. Spectral karyotyping unambiguously identified and classified the origin of the additional chromosomal material as being derived from chromosome 4. To determine the limit of sensitivity of spectral karyotyping, a case with a submicroscopic translocation (unrecognizable in both metaphase and prometaphase chromosomes) involving chromosomes 16 and 17 was examined. This t(16;17) had been previously demonstrated by FISH with cosmid probes and the reciprocal interchange of chromatin estimated at approximately 500 kbp. Spectral karyotyping with metaphase chromosomes from this patient failed to identify the known t(16;17) suggesting that the limit of sensitivity for metaphase chromosome analysis with currently available painting probes to be between 500-1,500 kbp.
To demonstrate that spectral karyotyping is an approach that can be used to complement conventional banding analysis, hybridization on previously G-banded chromosomes was performed. Probably due to the trypsin digestion that is required for G-banding, the signal intensity was slightly reduced as compared to metaphases that were not previously G-banded. The loss of signal intensity was approximately 10%, and could therefore easily be compensated for by prolonged exposure times. A slightly increased noise at the edges of previously G-banded chromosomes compared to non G-banded chromosomes was also observed. However, the classification of the metaphase could be readily achieved.
Thus, according to the above described approach, first, chromosomes of a single normal karyotype are identified by a conventional chromosome banding technique (e.g., G-banding or R-banding). Second, the same chromosomes are hybridized with chromosome paints as described above. Third, the average spectrum characterizing pixels attributed to any chromosome type (e.g., 1-22, X and Y in human male) as previously determined by chromosome banding is calculated and the resulting spectra (e.g., 24 for human) form a library of reference spectra. Fourth, the reference spectra arc thereafter used for classification of pixels of new karyotypes into their chromosomes.
The above described results and results by others, see, for example, Speicher et al. (1996) Nature Genetics. 12:368-375, were accomplished by blocking the highly repetitive sequences in the chromosomal paints with excess of unlabeled Cot-I fraction (GIBCO-BRL Life-Technologies, Scotland).
DENHYB.TM. (DENHYB is a trademark of Ingen Laboratories Inc.) solutions are designed to be used for the suspension or dilution of investigator-developed or commercially available DNA probes. DENHYB.TM. solutions are developed to maximize denaturation and hybridization between DNA probes and target DNAs in cellular samples. DENHYB.TM. solutions are compatible with directly and indirectly labeled DNA probes. Two DENHYB.TM. solutions are available from Sigma depending on the nature of targets or probes:
DENHYB.TM. (Cat. No. D001): For FISH with repeat sequence (satellite), painting, and also unique sequence probes.
DENHYB.TM. (Cat. No. D003): Recommended for FISH with unique sequence probes.
DENHYB.TM. solutions are recommended for use with blocking DNA when both the probe and target include repetitive sequences.
Using DENHYB.TM. solutions enables to dilute the probes by 10 to 200-fold, depending on the specific application and probe employed, while retaining maximum detection sensitivity. DENHYB.TM. solutions also reduce hybridization times to between 15 minutes to accomplish FISH on interphase nuclei and metaphase cells with satellite probes. For FISH with paint or unique sequence probes, hybridization for about 3-15 hours is sufficient. Usin DENHYB.TM., therefore, reduces costs by a large factor.
There is thus a widely recognized need for, and it would be highly advantageous to have a method of and a composite for fluorescent in situ hybridization using a hybridization composite of chromosomal paints which is devoid of added unlabeled highly repetitive DNA sequences.